Application of biological agent related to StERF1A gene in improving resistance of potato to bacterial wilt

Abstract

The present application relates to the technical field of plant breeding, in particular to StERF1A Application of a gene related biological agent in improving the resistance of potato to bacterial wilt, the biological agent StERF1A The nucleotide sequence of the gene is shown as SEQ ID NO. 1. The present application discloses for the first time StERF1A As a negative regulatory factor involved in the mechanism of potato resistance to bacterial wilt: overexpression StERF1A Can increase the accumulation amount of bacterial wilt in leaves, inhibit the expression of disease resistance related genes Pto 、 PR1b1 And PR1a The content of active oxygen ROS and the activity of related enzymes SOD, POD and CAT are reduced; and the expression of down-regulation StERF1A The opposite effect is presented, which significantly enhances the resistance of potato to bacterial wilt. The present application provides an important gene target and theoretical basis for breeding new potato varieties with high resistance to bacterial wilt.

Description

Technical Field

[0001] This invention relates to the field of plant breeding technology, specifically to StERF1A Application of gene-related biological agents in improving resistance to bacterial wilt in potatoes. Background Technology

[0002] potato( Solanum tuberosum L. (Ralstonia solanacearum) is the fourth largest food crop, playing a vital role in agricultural production and food security. It is caused by Ralstonia solanacearum (Ralstonia solanacearum). Ralstonia solanacearum Bacterial wilt caused by *Ralstonia solanacearum* is one of the most devastating soil-borne diseases in potato production. This pathogen infects the vascular system of the plant, leading to leaf wilting, stem browning, and ultimately, the death of the entire plant. Due to the wide host range and rapid genetic variation of *Ralstonia solanacearum*, and the lack of effective chemical control methods, breeding resistant varieties has become the fundamental strategy for controlling this disease. However, existing potato germplasm resources lack genes resistant to bacterial wilt, and traditional breeding methods are time-consuming and inefficient.

[0003] Therefore, it is urgent to develop a new strategy to improve resistance to bacterial wilt in potatoes by discovering novel disease-resistant genes and analyzing their regulatory mechanisms. Summary of the Invention

[0004] To solve the above problems, the present invention provides StERF1A Application of gene-related biological agents in improving resistance to bacterial wilt in potatoes.

[0005] This invention is achieved through the following technical solution: StERF1A The application of gene-related biological agents in improving resistance to bacterial wilt in potatoes, the aforementioned StERF1A The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0006] SEQ ID NO.1: ATGTTTGAAAATATAGATTTTGAAAATGATTATGCCCTTCTTGAATCAATTAAGCTTCAATTACTTGAAGATTGGGAATGGGAAAATCCTGTAACAAGCTCAGATAATTCAACCTCAACTTATAGCCGAAACAATAGTATTGAGTCTAATAATTCCTTATCGAATGATTTTGATTATTCAACTGACAAATTTCTTTCCGATTTGCTTAATGATAATGACGTTGGATATGGATCGGATCCTGTAATCCCAAATGTGAAATCGGAGCCTGAAATTTGGAATTTTGCGGAATTTGCGGCGGCGACGCAGTATACGACGGCGGAGGTGAGAGTGGAACCACCACCACAACTTGTAGTGACAGCGCCGCCACAGACGCTGCCGACGGCGAGGCACTATAGAGGTGTGAGACAGAGACCTTGGGGAAAATTTGCGGCGGAAATTAGGGATCCGGCGAAAAATGGGCAAAGAGTGTGGTTGGGTACGTATGAGACGGCGGAGGACGCAGCGTTTGCTTATGATAAAGCGGCGTTTCGCATGAGGGGTTCACGTGCAATGCTGAATTTCCCACTGAGGATTAATTCTGGTGAGCCGGAACCCATTAGAGTCAGGTCGAAGAAGTCATCAATGTCGCCGGAATGTTCTTCTTCGTCGTCATCGGATAATGCGCCGGGGAAGAGGAGGAAGAAGGTCCCTCAAGTGGTATAA。

[0007] Preferably, the StERF1A amino acid sequence of the protein expressed by the gene is as shown in SEQ ID NO.2.

[0008] SEQ ID NO.2: MFENIDFENDYALLESIKLQLLEDWEWENPVTSSDNSTSTYSRNNSIESNNSLSNDFDYSTDKFLSDLLNDNDVGYGSDPVIPNVKSEPEIWNFAEFAAATQYTTAEVRVEPPPQLV VTAPPQTLPTARHYRGVRQRPWGKFAAEIRDPAKNGQRVWLGTYETAEDAAFAYDKAAFRMRGSRAMLNFPLRINSGEPEPIRVRSKKSSMSPECSSSSSSDNAPGKRRKKVPQVV.

[0009] Preferably, the biological agent is StERF1A Gene recombinant silencing vectors or recombinant engineered bacteria.

[0010] Preferably, the recombinant silencing vector is obtained by ligating double-stranded RNA to a backbone plasmid.

[0011] Preferably, the double-stranded RNA comprises a long fragment sequence with nucleotide sequences as shown in SEQ ID NO. 11 and a short fragment sequence with nucleotide sequences as shown in SEQ ID NO. 12.

[0012] Preferably, the backbone plasmid is pBI121.

[0013] Preferably, the recombinant Agrobacterium is obtained by introducing the recombinant silencing vector into Agrobacterium.

[0014] Preferably, the Agrobacterium is GV3101.

[0015] Preferably, the recombinant silencing vector is used to reduce StERF1A To increase the expression level of [a specific substance], thereby improving resistance to bacterial wilt in potatoes.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention provides StERF1A The application of gene-related biological agents in improving resistance to bacterial wilt in potatoes, the aforementioned StERF1A The nucleotide sequence of the gene is shown in SEQ ID NO.1. This invention discloses for the first time... StERF1A It acts as a negative regulator in the mechanism of resistance to bacterial wilt in potatoes. Experiments have shown that inoculation with Ralstonia solanacearum... StERF1A The expression level increased significantly, and overexpression was observed. StERF1A It can increase the accumulation of Ralstonia solanacearum, while lowering... StERF1A This reduces the accumulation of Ralstonia solanacearum, indicating that the ERF transcription factor StERF1A is an endogenous pathogenic factor in potatoes. StERF1A The process of gene negative regulation of resistance to Ralstonia solanacearum in potatoes. StERF1A Genes are ubiquitous in plants; RNAi technology can be used to downregulate them in potatoes. StERF1A , obtained StERF1A Down-regulated plants exhibited significantly higher resistance to Ralstonia solanacearum than wild-type plants. Therefore, by down-regulating / knocking out... StERF1A It is expected to significantly improve the disease resistance and yield of potatoes. The analysis of the function and mechanism of action of ERF1A provides a theoretical and methodological basis for obtaining new crop varieties with high resistance and high yield, and therefore has good practical application value. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The present invention relates to potatoes inoculated with Ralstonia solanacearum. StERF1A Graph showing changes in expression levels.

[0019] Figure 2 In the transgenic lines of this invention StERF1A The expression level detection graph.

[0020] Figure 3 For the present invention StERF1A Graph showing the effect of Ralstonia solanacearum accumulation in potatoes; Figure 3 In the diagram, A shows the phenotypic results after inoculation with Ralstonia solanacearum; B shows the pathogen marker genes after inoculation with Ralstonia solanacearum. Flic Quantitative detection results graph.

[0021] Figure 4 This is a graph showing the detection of ROS levels, SOD enzyme activity, POD enzyme activity and CAT enzyme activity of different strains after infection with Ralstonia solanacearum according to the present invention. Figure 4 In the diagram, A represents the ROS content detection graph; B represents the SOD enzyme activity detection graph; C represents the POD enzyme activity detection graph; and D represents the CAT enzyme activity detection graph.

[0022] Figure 5 Different strains of Ralstonia solanacearum infected with this invention Pto , PR1b1 and PR1a Gene expression level detection graph; Figure 5 In the middle, A is Pto Gene expression level detection graph; B is PR1a Gene expression level detection graph; C represents PR1b1 Gene expression level detection graph. Detailed Implementation

[0023] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0024] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0025] The beneficial effects of the present invention will be illustrated below through specific embodiments.

[0026] The potatoes used in this invention are Xisen No. 6 potatoes, supplied by Leling Xisen Potato Group Co., Ltd. The *Ralstonia solanacearum* fungus is... Ralstonia solanacearum Purchased from the China General Microbiological Culture Collection Center, with the number CGMCC1.12711.

[0027] All reagents, consumables, and culture media used in this invention are common commercially available products.

[0028] In this invention, all primer sequences are oriented from the 5' to the 3' end.

[0029] Example 1: Potato StERF1A Expression induced by Ralstonia solanacearum Ralstonia solanacearum stored at -80℃ was inoculated onto TTC solid medium and cultured at 28℃ for 2 days. Clones were then picked, activated, and resuspended in 10 mM MgCl2 solution to OD. 600 =1.0, root irrigation method for infecting wild-type potatoes.

[0030] Samples were taken at specific time points after infection, and total RNA was extracted from the plants. cDNA was synthesized via reverse transcription. The primer sequences used were forward primer F and reverse primer R. The nucleotide sequence of the forward primer F is shown in SEQ ID NO. 3: AATGGGCAAAGAGTGTGG; the nucleotide sequence of the reverse primer R is shown in SEQ ID NO. 4: CGATGACGACGAAGAAGAAC. qRT-PCR detection was performed. StERF1A Changes in expression levels showed that Ralstonia solanacearum infection could induce... StERF1A Expressions, such as Figure 1 As shown.

[0031] Example 2 1. StERF1AConstruction of recombinant overexpression vector: Overexpression refers to the process of significantly increasing the expression level of a target gene in cells by increasing the copy number of the target gene or improving its transcription efficiency. The overexpression vector used in this invention is pBI121. First, cDNA was extracted from Xisen 6 potato, and primers were used... StERF1A -F and StERF1A PCR amplification was performed using the -R method to obtain the target fragment. Subsequently, the fragment and the pBI121 vector were simultaneously digested using a specific restriction enzyme. The target fragment and the pBI121 vector were ligated using T4 ligase. After successful ligation, the fragment was transformed into Agrobacterium GV3101 competent cells to obtain... StERF1A Overexpression vector.

[0032] StERF1A The nucleotide sequence of -F is shown in SEQ ID NO. 5: TCTAGAATTGTTTGAAAATATAG.

[0033] StERF1A The nucleotide sequence of -R is shown in SEQ ID NO. 6: GAGCTTTATCCACTTGAGG.

[0034] 2. StERF1A Construction of recombinant silencing vectors: RNA interference (RNAi) is a gene silencing mechanism mediated by double-stranded RNA that can efficiently and specifically degrade homologous mRNA. This invention selects... StERF1A Primers were designed around the 300bp sequence near the 5' end of the cDNA to ensure high specificity for this sequence in potatoes. Double-stranded RNA is abbreviated as dsRNA.

[0035] First, cDNA was extracted from the Hissen 6 potato and then PCR was performed using primers. StERF1A -F1 and StERF1A -R1 amplifies a long sequence containing dsRNA and stem-loop sequences. Restriction endonucleases are used. Xba I and Sma I. The amplified fragment and the plant expression vector pBI121 were double-digested with enzymes, ligated with T4 DNA ligase, and then transformed into E. coli DH5α competent cells. The recombinant intermediate vector was obtained by screening and named pBI121-. StERF1A -long.

[0036] Furthermore, using the same cDNA as a template, primers were used... StERF1A -F2 and StERF1A -R2 amplifies a short fragment sequence, which is a dsRNA sequence without stem-loop structure. (The text then repeats the process.) Sac I and Sma I. Short fragments and the aforementioned intermediate vector pBI121- StERF1A -long is double-digested, and the digested products are ligated with T4 DNA ligase and then transformed into Agrobacterium GV3101 competent cells to finally obtain an RNAi expression vector for plant transformation, namely a recombinant silencing vector.

[0037] StERF1A The nucleotide sequence of -F1 is shown in SEQ ID NO. 7: TCTAGACTTGAAGATTGGGAATG.

[0038] StERF1A The nucleotide sequence of -R1 is shown in SEQ ID NO. 8: CCCGGGCAACCACACTCTTTGC.

[0039] StERF1A The nucleotide sequence of -F2 is shown in SEQ ID NO. 9: GAGCTCCTTGAAGATTGGGAATG.

[0040] StERF1A The nucleotide sequence of -R2 is shown in SEQ ID NO. 10: CCCGGGCACCTCCGCCGTC.

[0041] The nucleotide sequence of the long fragment is shown in SEQ ID NO. 11, and is as follows: CTTGAAGATTGGGAATGGGAAAATCCTGTAACAAGCTCAGATAATTCAACCTCAACTTATAGCCGAAACAATAGTATTGAGTCTAATAATTCCTTATCGAATGATTTTGATTATTCAACTGACAAATTTCTTTCCGATTTGCTTAATGATAATGACGTTGGATATGGATCGGATCCTGTAATCCCAAATGTGAAATCGGAGCCTGA AATTTGGAATTTTGCGGAATTTGCGGCGGCGACGCAGTATACGACGGCGGAGGTGAGAGTGGAACCACCACAACTTGTAGTGACAGCGCCGCCACAGACGCTGCCGACGGCGAGGCACTATAGAGGTGTGAGACAGAGACCTTGGGGAAAATTTGCGGCGGAAATTAGGGATCCGGCGAAAAATGGGCAAAGAGTGTGGTTG.

[0042] The nucleotide sequence of the short fragment is shown in SEQ ID NO. 12, and is as follows: CTTGAAGATTGGGAATGGGAAAATCCTGTAACAAGCTCAGATAATTCAACCTCAACTTATAGCCGAAACAATAGTATTGAGTCTAATAATTCCTTATCGAATGATTTTGATTATTCAACTGACAAATTTCT TTCCGATTTGCTTAATGATAATGACGTTGGATATGGATCGGATCCTGTAATCCCAAATGTGAAATCGGAGCCTGAAATTTGGAATTTTGCGGAATTTGCGGCGGCGACGCAGTATACGACGGCGGAGGTG.

[0043] By building StERF1A Using overexpression and RNAi vectors, transgenic potato lines, including the overexpressing line StERF1A and the downregulated line StERF1Ai, were obtained through tissue culture techniques. The steps for cultivating transgenic plants are as follows: Prepare potatoes that have grown to 5 branches, cut stem segments and allow them to dedifferentiate for 3 days. Infect the callus tissue with Agrobacterium containing a target gene overexpression vector or a target gene RNAi expression vector for 20 minutes. The OD of the infection solution is measured. 600 The value was 0.5, and the infection time was 20 minutes. After drying the stem segments on filter paper, they were transferred to MSA medium containing 0.2 mg / L naphthaleneacetic acid, 0.02 mg / L gibberellin, and 2.5 mg / L zeatin nucleoside, and cultured in the dark for 2 days. The segments were then washed three times with sterile water containing 1‰ termethin, followed by three washes with MS solution containing 1‰ termethin. After drying, they were transferred to MSA medium containing 1‰ termethin and cultured for 10 days. Afterward, they were transferred to selective medium until budding. The selective medium was MS medium containing 0.02 mg / L naphthaleneacetic acid, 0.02 mg / L gibberellin, 2 mg / L zeatin nucleoside, 1‰ termethin, and 50 μg / mL kanamycin. The buds were then transferred to rooting medium to obtain transgenic plants. The rooting medium was MS medium containing 1‰ termethin and 50 μg / mL kanamycin.

[0044] StERF1A Relative expression levels, such as Figure 2As shown, this demonstrates the successful acquisition of the overexpression line StERF1A and the downregulated line StERF1Ai. In the figure, wild type represents untransgenic wild-type potato plants; StERF1A-1, StERF1A-2, and StERF1A-3 represent overexpression lines; and StERF1Ai-1, StERF1Ai-2, and StERF1Ai-3 represent downregulated lines.

[0045] Example 3 The overexpressing strain StERF1A-1 and the downregulated strain StERF1Ai-1 were selected as the experimental groups. The overexpressing strain StERF1A-1 is represented by StERF1A in the figure; the downregulated strain StERF1Ai-1 is represented by StERF1Ai in the figure. The wild type was used as the control group, represented by wild type in the figure. Phenotypic observation and pathogen marker genes were determined after inoculation with *Ralstonia solanacearum*. Flic Quantitative detection results showed overexpression StERF1A Increased the accumulation of Ralstonia solanacearum, lowered StERF1A Expression will reduce the accumulation of Ralstonia solanacearum, such as Figure 3 As shown. Therefore, it indicates StERF1A It may be an endogenous pathogenic factor present in the plant's disease resistance process.

[0046] Example 4 StERF1A Downregulation can increase ROS levels and the activity of SOD, POD, and CAT. The overexpressing strain StERF1A-1 and the downregulated strain StERF1Ai-1 were selected as the experimental groups; the wild-type strain was used as the control group. The overexpressing strain StERF1A-1 was represented by StERF1A in the figure; the downregulated strain StERF1Ai-1 was represented by StERF1Ai in the figure.

[0047] ROS, as a signaling molecule, plays a role in disease resistance, and rapid ROS synthesis is a key indicator of plant defense system activation. Since ROS formation and enzyme activity should be determined throughout the entire timeframe, this invention involves harvesting leaves at 0 dpi, 1 dpi, 3 dpi, and 5 dpi after inoculation with *Ralstonia solanacearum*, and measuring ROS levels, SOD enzyme activity, POD enzyme activity, and CAT enzyme activity in wild-type lines, the overexpressing line *StERF1A-1*, and the downregulated line *StERF1Ai-1*, respectively.

[0048] The results are as follows Figure 4As shown, after inoculation with Ralstonia solanacearum, the ROS content, SOD activity, POD activity, and CAT activity of the control line, the overexpressing line StERF1A-1, and the downregulated line StERF1Ai-1 surged one day after inoculation. At 3 dp and 5 dpi, the ROS content, SOD activity, POD activity, and CAT activity of the downregulated line StERF1Ai-1 were significantly higher than those of the control line, while those of the overexpressing line StERF1A-1 were lower than those of the control line.

[0049] Example 5 StERF1A Overexpression can suppress the expression of key genes in plant pathogenesis. The overexpressing strain StERF1A-1 and the downregulated strain StERF1Ai-1 were selected as the experimental groups; the wild-type strain was used as the control group. The overexpressing strain StERF1A-1 was represented by StERF1A in the figure; the downregulated strain StERF1Ai-1 was represented by StERF1Ai in the figure.

[0050] This invention uses qRT-PCR to identify StERF1A With defense genes Pto The regulatory relationship between PRs and [other substances]. Results are as follows: Figure 5 As shown, compared with the control group, the overexpression of the StERF1A-1 strain... Pto , PR1b1 and PR1a Gene expression levels were significantly decreased, while those in the downregulated line StERF1Ai-1 were significantly increased. Therefore, this result indicates that... StERF1A It may negatively regulate potato plants Pto The expression of PRs genes was used to increase the susceptibility of potatoes to Ralstonia solanacearum.

[0051] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0052] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. StERF1A The application of gene-related biological agents in improving resistance to bacterial wilt in potatoes is characterized by, The StERF1A The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The StERF1A The amino acid sequence of the protein expressed by the gene is shown in SEQ ID NO.

2.

3. The application according to claim 1, characterized in that, The biological agent is StERF1A Gene recombinant silencing vectors or recombinant engineered bacteria.

4. The application according to claim 3, characterized in that, The recombinant silencing vector is obtained by ligating double-stranded RNA to a backbone plasmid.

5. The application according to claim 4, characterized in that, The double-stranded RNA comprises a long fragment sequence as shown in SEQ ID NO. 11 and a short fragment sequence as shown in SEQ ID NO.

12.

6. The application according to claim 4, characterized in that, The backbone plasmid is pBI121.

7. The application according to claim 3, characterized in that, The recombinant Agrobacterium was obtained by introducing the recombinant silencing vector into Agrobacterium.

8. The application according to claim 7, characterized in that, The Agrobacterium species in question is GV3101.

9. The application according to claim 3, characterized in that, Using recombinant silencing vectors to reduce StERF1A To increase the expression level of [a specific substance], thereby improving resistance to bacterial wilt in potatoes.

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